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Novel tumor wilting matter 2 ligand gene, its expressed tumor wilting matter and its preparation method

A technology of apoptin and tumor, applied in the field of medical bioengineering, can solve the problems of difficult and unstable refolding process, achieve the effect of simple preparation method, increase yield and quality

Inactive Publication Date: 2002-09-04
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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Problems solved by technology

However, due to prokaryotic recombinant expression of Apo-2L 114 When using a commercial expression vector, such as pET (Novagen) or pEQ (Qiagen), the recombinant protein is in the form of an inclusion body, which must undergo renaturation before it can exhibit biological activity, and the renaturation process is relatively Difficult and unstable, and not all inclusion bodies are reversible

Method used

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  • Novel tumor wilting matter 2 ligand gene, its expressed tumor wilting matter and its preparation method
  • Novel tumor wilting matter 2 ligand gene, its expressed tumor wilting matter and its preparation method
  • Novel tumor wilting matter 2 ligand gene, its expressed tumor wilting matter and its preparation method

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specific Embodiment approach 1

[0027] The specific embodiment one, set up cDNA library, screen Apo-2L I gene

[0028] 1. Preparation of total RNA from Chinese peripheral blood mononuclear cells

[0029] Isolate Chinese peripheral blood lymphocytes with lymphocyte separation medium according to routine, and culture them with RPMI-1640 medium (GIBCO Company), add 10% newborn calf serum (Hangzhou Sijiqing Biological Company), penicillin and streptomycin until adherent , mononuclear cells were obtained, and then stimulated with 10 μg / L endotoxin of Escherichia coli R595 (Department of Biochemistry and Molecular Biology, Basic Department, Second Military Medical University, Chinese People's Liberation Army) for 2 hours to activate mononuclear cells, and collected 10 7 Cells, total RNA was extracted with a total RNA extraction kit (Qiagen Company) to obtain total RNA of mononuclear cells.

[0030] 2. Establish cDNA library and screen Apo-2L I gene

[0031] According to the routine, the total RNA obtained above ...

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Abstract

The present invention relates to the field of medicine bio-engineering technology, and is a novel tumor wilting extract 2 ligand complete gene, i.e. Apo-2LI gene, and utilizes the said gene to prepare human soluble tumor wilting extract protein, i.e. tumor wilting extract or called Apo-2L I100. This invention uses Apo-2LI gene as template, and uses pBV220 to constitute prokaryotic expression vector pBV-Apo-2L I100, and its prepared Apo-2LI100 can directly express biological activity.

Description

technical field [0001] The invention relates to the technical field of medical bioengineering, and is a novel tumor apoptin 2 ligand gene obtained from Chinese peripheral blood mononuclear cells, and uses the gene to prepare human soluble tumor apoptosis protein—tumor apoptosis prime method. Background technique [0002] In 1995, foreign reports screened a gene encoding an anti-tumor protein from a human expressed sequence tag (EST) (GenBank No.: U57059; U37518; NM003810; XM 045049). The protein encoded by the gene is called Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), also known as tumor necrosis factor-related apoptosis-inducing ligand (Apo-2L) One of the necrosis factor (TNF) family members, it can effectively induce apoptosis of tumor and transformed cells, but has no effect on normal cells. The coding sequence of the human Apo-2L gene has 843 nucleotides, and the nucleotide sequence is shown in GenBank No.: U57059; U37518; NM 003810; XM 045049, and...

Claims

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Application Information

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IPC IPC(8): A61K38/17C07H21/04C07K14/435C12N15/12C12N15/63
Inventor 王梁华焦炳华
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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